Chemotherapeutic agent-incorporated pharmaceutical preparation

ABSTRACT

An apparatus and a method of remote control which can enable real time operation of a device in home from a terminal device at remote location through a network are provided. A first server communicates with a terminal device through the Internet and generates device control data for controlling the device. A second server communicates with the device in a predetermined manner to acquire and store a specific address of the device, generates transmission data for transmission of the received device control data from the first server to the device based on the specific address, and transmits the transmission data to the device. This allows real time control of the device from the terminal device through the network to be realized.

This application is the US national phase of international applicationPCT/JP2003/013860 filed 29, Oct. 2003, which designated the U.S. andclaims priority of JP 2002-320577, filed 1, Nov. 2002, the entirecontents of each of which are hereby incorporated by reference.

TECHNICAL FIELD

The present invention relates to a pharmaceutical preparation used intransfection of cells or living organisms with a chemotherapeutic agentby a gene delivery vector.

BACKGROUND ART

It is said that the cure rate in cancer treatment is about 50%. atpresent, and generally, such cure is brought about often by topicaltherapy such as surgical therapy and radiotherapy. It is in a very lowrate that chemotherapy as systemic therapy can contribute solely to cue,particularly in treatment of solid tumor, and usually chemotherapy isused in combination with various therapies.

On the other hand, surgical therapy enables surgery in every organcancer and is considered to reach completion as therapy, and no furtherimprovement in cure rate can be expected. The treatment results ofsusceptible organ cancers by radiotherapy also arrive at an almost fixedrate, and no further improvement in cure rate can be expected as well.

Accordingly, no significant improvement in cancer cure rate by thesetherapies can be expected in the future, and the development of furtherexcellent chemotherapy is essential for further improving the cancercure rate of 50% at present to arrive at cure for cancer.

An purpose of an anticancer agent used in chemotherapy lies in cytocidaleffect on cells having a high ability to grow, such as cancer cells, andits damage to normal cells particularly myeloid cells having a highcellular growth ability is significant, and as a result, severe pain isgiven to patients. This is because the transfer of the anticancer agentis due to systemic administration by an injection, and the anticanceragent reaches normal cells other than cancer cells, so that the normalcells are killed and homeostasis does not function.

At present, however, the effect of an anticancer agent administeredalone is regarded to be approximately about 30%, and it is expected thatgenetic information analysis and study on genome proceed so thatselection of a suitable anticancer agent feasible can be expected in thefuture, but it is said that the therapy with the anticancer agent atpresent results in higher side effects.

This is because normal cells are damaged by systemic administration ofan anticancer agent. Accordingly, if cancer tissue-specific delivery ofthe anticancer agent and subsequent incorporation thereof into cancercells can be established, an ideal system of delivering the anticanceragent can be realized. In addition, if incorporation of the anticanceragent into a vesicle is feasible, a therapeutic method that is specificfor target organ and cell with less influence (side effect) on normalcells can be established. Further, this can lead to reassessment ofanticancer agents whose development was abandoned because of theirstrong side effects.

DISCLOSURE OF THE INVENTION

As a result of extensive study for solving the problem described above,the present inventors could complete a pharmaceutical preparationcomprising, as an active ingredient, a virus envelope vector having achemotherapeutic agent incorporated therein.

Accordingly, the present invention provides, for example, apharmaceutical preparation comprising an anticancer agent or the likeincorporated in e.g. an inactivated HVJ-E vector having an ability toincorporate a foreign gene.

The present invention relates to a pharmaceutical preparation used intransfection of cells or living organisms with a chemotherapeutic agent,preferably a cancerocidal agent, an anticancer agent, or an antitumoragent (hereinafter referred to collectively as anticancer agent), byusing a gene delivery vector. The present invention relates morespecifically to a pharmaceutical preparation by which a highly toxicanticancer agent is transferred by a gene delivery vector to the livingorganism and allowed to reach a target organ or cell safely with areduction in side effects.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention is described in more detail.

The chemotherapeutic agent used in the present invention is notparticularly limited insofar as it is a low-molecular compound actingdirectly on cells, and for example, “Seikagaku Jiten” (Dictionary ofBiochemistry), third edition, published by Tokyo Kagaku Dojin,describes: “At present, the subject of chemotherapy, that is, therapyusing chemical substances of highly selective toxicity, has spread notonly to microbial infections but also to malignant tumors”, and it goeswithout saying that antibacterial agents, antitumor agents etc. arecontained in the chemotherapeutic agent.

Preferable examples of the chemotherapeutic agent in the presentinvention include a cancerocidal agent, an anticancer agent or anantitumor agent (hereinafter referred to collectively as anticanceragent), and specifically the anticancer agent includes bleomycin andderivatives thereof, anthraquinone-based cancerocidal agents includingadriamycin and daunomycin, mitomycin and derivatives thereof,actinomycin and derivatives thereof, taxane derivatives such as taxol,camptothecin and derivatives thereof such as irinotecan, cisplatin andderivatives thereof, staurosporine and derivatives thereof, vincristine,streptozotocin, 5-fluorouracil (5-FU) and derivatives thereof,viralbicin and dolastatin, as well as pharmacologically acceptable saltsthereof.

Preferable among these chemotherapeutic agents are bleomycin andderivatives thereof, and specific examples include bleomycin orpharmacologically acceptable salts thereof, or peplomycin orpharmacologically acceptable salts, more specifically bleomycinhydrochloride, bleomycin sulfate and peplomycin sulfate.

When the pharmaceutical preparation of the present invention is used asan anticancer agent, the type of cancer to which it is applicable is notparticularly limited, and specifically, solid cancer, blood cell cancer,and the like can be exemplified. Among these cancers, the solid canceris a preferable subject to which the pharmaceutical preparation isapplicable.

Specific examples of the solid cancer include lung cancer, breastcancer, digestive organ cancer, head and neck cancer, gynecologiccancer, urologic cancer, soft tissue and bone sarcoma, malignantlymphoma, cancers of unknown primary etc., and more specifically, fordigestive organ cancers, stomach cancer, colon cancer, and esophaguscancer are exemplified, for head and neck cancers, upper jaw cancer,tongue cancer, lip cancer, pharynx cancer, larynx cancer, and oralcavity cancer are exemplified, for gynecologic cancers, uterus cancer,ovarian cancer, and uterine cervical cancer are exemplified, and forurologic cancers, prostate cancer is exemplified.

Among these solid cancers, more preferable subjects include skin cancer,skin malignant tumor, head and neck cancers (upper jaw cancer, tonguecancer, lip cancer, pharynx cancer, oral cavity cancer, and the like),lung cancer (particularly primary and metastasis flat epithelialcancer), esophagus cancer, malignant lymphomas (reticulum sarcoma,lymphosarcoma, Hodgkin's disease, and the like), uterine cervicalcancer, neuroglioma, thyroid cancer, and prostate cancer.

The virus envelope vector in the present invention is a virus envelopewhich is prepared by removing RNA or DNA from virus. It is usuallyutilized to incorporate a gene, polynucleotide, oligonucleotide, plasmidor the like thereinto, for transfection.

The type of the virus is not limited, and specifically the virusincludes, for example, viruses belonging to a family selected from thegroup consisting of the retrovirus family, togavirus family, coronavirusfamily, flavivirus family, paramyxovirus family, orthomyxovirus family,bunyavirus family, rhabdovirus family, poxvirus family, herpes virusfamily, baculovirus family, and hepadna virus family.

Specifically, the virus in the present invention includes, for example,Sendai virus, retrovirus, adenovirus, adeno-associated virus, herpesvirus, vaccinia virus, poxvirus, influenza virus, and the like.

Among these viruses, Sendai virus (hemagglutinating virus of Japan(referred to hereinafter as HVJ)) that is one of mouse pneumonia virusescan be mentioned as a preferable example.

Specifically, Sendai virus, for example, VR-105 and VR-907 can bepurchased from American Type Culture Collection ((ATCC), telephone1-703-365-2700, P.O. Box 1549, Manassas, Va. 20108, USA,

-   on the World Wide Web at    atcc.org/SearchCatalogs/longview.cfm?view=av,152    376,VR-105&text=Sendai&max=20,-   on the World Wide Web at    atcc.org/SearchCatalogs/longview.cfm?view=av,137    5478,VR-907&text=Sendai&max=20).

The virus envelope vector is described in more detail in, for example,JP-A 2001-286282 (WO01/57204), JP-A 2002-065278, WO-A 03/014338(PCT/JP02/07879), and can be prepared specifically according to e.g.Example 8 in JP-A 2001-286282.

In the step of incorporating a chemotherapeutic agent in the virusenvelope vector, a surfactant is preferably used, and specific examplesof the surfactant include Triton X100, deoxycholic acid or a saltthereof, and cholic acid or a salt thereof. Preferably, the salt ofdeoxycholic acid includes sodium deoxycholate, and preferably, the saltof cholic acid includes sodium cholate.

The form of the pharmaceutical preparation of the present invention isnot limited, but its specific example is an injectbale solution,ointment, or the like, preferably an injectable solution.

Here, the present inventable solution is described in more detail byreference to the inactivated Sendai virus envelope vector (referred tohereinafter as HVJ-E vector).

When an anticancer agent is incorporated into the HVJ-E vector, theanticancer agent is dissolved in a buffer solution. The buffer solutionused herein is not limited, and specifically, for example, TE buffersolution (10 mM Tris, 1 mM EDTA (pH 8.0)), PBS (phosphate buffersolution) can be suitably selected and used wherein the pH of the buffersolution is preferably 6 to 9.

A preferable feature of the present invention is that, in an in vitroexperiment, an anticancer agent having a strong side effect or toxicitycan be incorporated into HVJ-E vector to be delivered directly intocells without leakage of the anticancer agent into a culture solution.

In an in vivo animal experiment, not systemic administration but localadministration of the anticancer agent is feasible, and the anticanceragent can be efficiently delivered into solid-cancer cells only.

Humans can be treated by chemotherapy of administering the anticanceragent-incorporated HVJ-E vector solely, or by locally administering itinto progressive cancer patients whom the anticancer agent cannot beadministered, to attain cancer regression, and simultaneously usingradiotherapy and/or surgical treatment to achieve further excellentanticancer effects.

In an in vitro experiment, host cells are transfected with theanticancer agent-incorporated HVJ-E vector. As the procedure in thiscase, for example, a method of adding a solution of the anticanceragent-incorporated HJV-E vector to a medium for culturing the cells canbe used.

The transfection is performed for about 30 minutes to 48 hours when itis carried out at 37° C. Judgment of the effect is conducted preferablyby counting the number of viable cells or by WST assay (technique ofcounting viable cells; cell counting kit, Dojin Kagaku).

When the subject of an in vivo animal experiment is, for example, amouse, it is preferable that a normal mouse that is not animmune-deficient mouse is used where the cancer cell is an isograft, andthat a nude mouse or SCID mouse is used in the case of xenograft.

Cancer cells cultured in a Petri dish are transplanted subcutaneously toa mouse, and after growth of the transplanted cells, the anticanceragent-incorporated HVJ-E vector is administered into the grown solidcancer, and the major axis and minor axis of the cancer can be measuredto determine the anticancer effect.

According to the present invention, there is provided a method capableof delivering an anticancer agent having a strong side effect to thesite of a cancer easily and safely.

Accordingly, the HVJ-E vector can be used to enable new chemotherapy forany solid cancer, which is increasing rapidly in Japan, such as lungcancer, breast cancer, digestive organ cancers such as stomach cancer,colon cancer or esophagus cancer, head and neck cancers (upper jawcancer, tongue cancer, lip cancer, pharynx cancer, larynx cancer, oralcavity cancer etc.), gynecologic cancers (uterus cancer, ovarian cancer,uterine cervical cancer etc.), urologic cancers (prostate cancer), softtissue and bone sarcoma, malignant lymphoma, cancers of unknown primary,and the like.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graph wherein the numbers (mean±standard deviation) ofviable cells in the respective groups in an in vitro experiment arecompared.

FIG. 2 is a graph wherein the average tumor volumes (mean±standarddeviation) in the respective groups in an in vivo experiment arecompared.

FIG. 3 is a graph showing a change in the average tumor volume(mean±standard deviation) in each group, relative to the medium (PBS)group, on Day 16 after administration in an in vivo experiment.

FIG. 4 is a graph showing the result of tumor volume in Example 3.

EXAMPLES

Hereinafter, the present invention is described in more detail byreference to Examples, but the present invention is not limited by theseExamples.

Example 1 In vitro Experiment

According to Example 8 in JP-A 2001-286282, 6,000 HAU/600 μl (for six6-well plates) of inactivated HVJ-E vector was allowed to have a changein temperature from −80° C. to 34.5° C. A microtube containing thesample was centrifuged at 15,000 rpm for 15 minutes at 4° C. to give theHVJ-E vector as a precipitate, and the supernatant was removed. Theresulting precipitate was suspended in 60 μl bleomycin/PBS (5 mg/ml)(bleomycin HCl manufactured by Nippon Kayaku Co., Ltd.) solution.Further, 2 μl of 3% Triton-X100 was added thereto to prepare a samplecontaining Triton-X100 at a final concentration of 0.1%, which was thenleft on ice for 15 minutes. Thereafter, 500 μl PBS solution was addedthereto. The microtube was centrifuged at 15,000 rpm for 15 minutes at4° C., and the supernatant was removed without removing the precipitate,and 500 μl PBS solution was added again to the precipitate. Themicrotube was centrifuged again at 15,000 rpm for 15 minutes at 4° C.,and the supernatant was removed without removing the precipitate.

The resulting precipitate was suspended in 180 μl PBS, and the resultingsample solution was pipetted in a volume of 30 μl/microtube into 6microtubes. Five microlitters of protamine sulfate solution prepared at5 mg/ml and 500 μl DMEM solution (Dulbecco's modified Eagle medium) wereadded to each tube.

As the administration group, the followings were prepared and comparedto evaluate their effect.

-   HVJ-bleomycin group; 1,000 HAU, bleomycin 200 ng/DMEM 500 μl/well.-   HVJ-PBS group: 1,000 HAU/DMEM 500 μl/well.-   1/50 bleomycin group: bleomycin 50 μg/DMEM 500 μl/well.-   1/500 bleomycin group: bleomycin 5 μg/DMEM 500 μl/well.-   1/5,000 bleomycin group: bleomycin 500 ng/DMEM 500 μl/well.-   Medium group: DMEM

The above sample solution was added to mouse colon carcinoma cell CT26prepared on a 6-well plate. The plate was kept for 30 minutes in anincubator at 37° C., and then the medium was exchanged with 500 μlmedium (DMEM containing 10% FCS). The cells were incubated for 2 days ina CO₂ incubator at 37° C. Two days later, the number of viable cells wascounted to evaluate the anticancer effect.

The results are shown in the following Table and in FIG. 1.

TABLE 1 Number Standard Administration group of mice Mean deviation 1/50Bleomycin group 2 81800 16688 1/500 Bleomycin group 2 164600 138591/5,000 Bleomycin group 2 196800 15274 HVJ-bleomycin group 2 16800 170HVJ-PBS group 2 201100 8627 Medium group 2 220100 23617

The numbers of viable cells on average in the medium group and HVJ-PBSgroup were 220,100 and 201,100 respectively. The numbers of viable cellsin the groups given bleomycin added to the medium (500 ng, 5 μg, 50 μg)were 196,800, 164,600 and81,800 respectively, while the number of viablecells in the bleomycin HVJ-E incorporation group was 16,800. Inpercentage relative to the medium group (=100%), the degree of viablecells in the HVJ-PBS group was 91.4%, and the degrees of viable cells inthe groups given bleomycin (500 ng, 5 μg and 50 μg) were 89.4%, 74.8%and 33.9% respectively, while the degree of viable cells in thebleomycin HVJ-E incorporation group was as low as 7.6%.

As a result, the dramatic cytocidal effect could be achievedsuccessfully by incorporation of bleomycin into HVJ-E. The significanteffect of bleomycin introduced directly by the HVJ-E vector into cellscan be understood when it is taken into consideration that the cytocidaleffect of bleomycin added to the culture solution is not so high.

This result indicated that by incorporating, into HVJ-E vector, theanticancer agent causing a severe side effect upon systemicadministration, the chemical can be delivered directly to affected cellsof the patient.

Example 2 In vivo Experiment

HVJ-E vector (6,000 HAU/600 μl) was rapidly dissolved by shifting thetemperature from −80° C. to 34.5° C. A microtube containing the samplewas centrifuged at 15,000 rpm for 15 minutes at 4° C. to give the HVJ-Evector as a precipitate, and the supernatant was removed. The resultingprecipitate was suspended in 60 μl bleomycin/PBS (40 mg/ml) solution.Further, 2 μl of 3% Triton-X100 was added thereto to the finalconcentration of 0.1%, which was then kept on ice for 15 minutes.Thereafter, 500 μl PBS solution was added thereto. The microtube wascentrifuged at 15,000 rpm for 15 minutes at 4° C., and the supernatantwas removed without removing the precipitate, and 500 μl PBS solutionwas added thereto. The microtube was centrifuged again at 15,000 rpm for15 minutes at 4° C., and the supernatant was removed without removingthe precipitate. The resulting precipitate was suspended in 120 μl PBS.

The following administration groups were prepared and were compared toevaluate their effect.

-   HVJ-bleomycin administration group: 5,000 HAU, bleomycin 6.5 μg/100    μl/mouse-   HVJ-PBS administration group: 5,000 HAU/100 μl/mouse-   65 μg/ml bleomycin administration group: bleomycin/PBS (65 μg/ml),    100 μl/mouse-   PBS administration group: 100 μl PBS

In this animal experiment, BALB/c mice (8-week-old, male) were used. Thesite in which the cancer cell colon carcinoma CT26 was transplanted wasa subcutaneous region in the back of a mouse, and hair on the back wasshaved for measuring the volume of the transplanted cancer cells. TheCT-26 cells to be transplanted were suspended in DMEM medium containing10% FCS, and 5×10⁶ cells (100 μl PBS/mouse) were transplanted in theback. The mice were anesthetized by intraperitoneal administration of500 μl of 20-fold-diluted Nembutal injection. The volume of thetransplanted cancer cells was estimated by calculation of majoraxis×minor axis×minor axis/2. When the diameter of the tumor reached 7to 8 mm one week after transplantation, 100 μl of the sample preparedabove was administered into the site of the cancer in the mouse. On Days7, 10, 13, 16 and 19 (that is, 3-day intervals) after transplantation ofthe cancer cells, the tumor diameter was measured to evaluate theanticancer effect. The number of animals was 8 in each group.

The results are shown in the following table and in FIG. 2. (Upper,mean; lower, standard deviation)

TABLE 2 Number of days after administration 7 10 13 16 65 mg/mlbleomycin 158.4 413.70 754.7 1234.6 25.4 71.20 206.6 332.8 HVJ-bleomycin136.2 285.70 456.7 676.1 16.2 77.60 116.4 209.2 HVJ-PBS 164.3 362.20688.1 1083.1 23.8 73.70 143.7 243.8 Medium (PBS) 158.7 418.20 738.71277.7 33.3 62.50 97.9 162.7

The average tumor volume in each group on Day 16 after administration,and the rate of change relative to the medium (PBS) group are shown inFIG. 3.

In the tumor diameter with no difference recognized at the time ofinoculation with the prepared sample, a difference was recognized on andafter Day 3 (corresponding to Day 10 in FIG. 2) after administration ofthe sample (FIG. 2). On Day 9 (corresponding to Day 16 in FIG. 2, thatis, 9 days after administration of the sample), the volumes of thetumor, calculated by the equation above, were 1,277 mm3 in the PBSadministration group, 1,235 mm³ in the 65 μg/ml BLM administrationgroup, 1,083 mm³ in the HVJ-PBS administration group and 676 mm³ in theHVJ-BLM administration group respectively (FIG. 2). When expressed inpercentage, the PBS administration group: 65 μg/ml BLM administrationgroup: HVJ-PBS administration group: HVJ-BLM administrationgroup=0%:3.4%:15.2%:47.1% (FIG. 3). In the group with bleomycinadministered directly to the tumor-affected area, the tumor shrinkageeffect was as low as 3.4% relative to the effect (0%) of the PBSadministration group, and the shrinkage effect on tumor volume washardly recognized. Whether this is due to the direct administration intothe tumor as opposed to usually conducted systemic administration or dueto administration at low concentration, or due to another factor, cannotbe judged from the present results. The tumor volume shrinkage effect inthe HVJ-PBS administration group was 15.2%, thus indicating that eventhe HVJ-E vector only attains a certain effect. This anticancer effectat a certain extent is possibly due to the immune action induced byHVJ-E. On the other hand, the tumor volume in the incorporated bleomycinadministration group was 47.1%, indicating a high antitumor effect.

This Example has revealed:

-   -   Antitumor effect was hardly recognized in vivo by direct        administration of bleomycin to the solid tumor cells.    -   Even the HVJ-E vector only was recognized to attain a weak        antitumor effect.    -   When bleomycin is incorporated into HVJ-E and administered        directly into the solid tumor, an excellent antitumor effect was        recognized.

Example 3 In Vivo Experiment (2)

(1) Test Design

Mouse colon cancer-derived CT-26 cells were transplanted subcutaneouslyinto the backs of 8-week-old BALB/cAnNCrj male mice, and 0.2 or 0.4 mgPlatocin injection (cisplatin, CDDP)/body was once administeredintraperitoneally into the animals (10 animals in each group) whereinthe diameter of the tumor (major axis) reached about 5 mm, and on Day 1after administration, HVJ-E, or HVJ-E/BLM containing 13.2 mg bleomycin,was administered once to the tumor. On Day 21 after administration intothe tumor, the mice were sacrificed to examine the antitumor action ofHVJ-E/BLM.

The constitution of the group in this test is shown below.

Intraperitoneal Administration of administration of the test substancethe comparative control into the tumor Group substance (mg/body)*(mg/tumor)** Control group Physiological 0 Physiological 0 saline salineHVJ-E group Physiological 0 HVJ-E 0 saline  13 mg/tumor Physiological 0HVJ-E/BLM 13 HVJ-E/BLM group saline 0.2 mg/body CDDP CDDP 0.2Physiological 0 group saline 0.4 mg/body CDDP CDDP 0.4 Physiological 0group saline 0.2 mg/body CDDP 0.2 HVJ-E/BLM 13 CDDP-13 mg/tumorHVJ-E/BLM group 0.4 mg/body CDDP 0.4 HVJ-E 0 CDDP-HVJ-E group *amount ofcisplatin (CDDP) **amount of bleomycin (BLM)(2) Experimental Methods2-1) Culture of Cancer Cells

Mouse colon cancer-derived CT-26 cells were cultured in DMEM mediumcontaining 10% FBS at 37° C. in the presence of 5% CO₂.

The cells were cultured in a 75-cm ² flask. The cells upon becomingabout 80% confluent were subjected to subculture. After the DMEM(containing 10% FBS) was removed, the cells were washed with 10 mLphosphate-buffered physiological saline (PBS), and then the cells werereleased at 37° C. by adding 1 mL PBS containing 0.25% trypsin and 1mmol/L EDTA-2Na. After 9 mL DMEM medium was added, the cells werecollected and centrifuged (1000 rpm, 5 minutes), to recover the cells.After the supernatant was removed, the cells were diluted with DMEMmedium containing 10% FBS and then cultured.

2-2) Preparation of a Suspension of the Cancer Cells

After the culture was removed from the cells which had became about 80%confluent, the culture flask was washed with PBS. A small amount of PBScontaining 0.25% trypsin and 1 mmol/L EDTA-2Na was added and the flaskwas kept at 37° C. until release of the cells was initiated. The cellswere collected with DMEM medium and centrifuged (1000 rpm, 5 minutes).After removal of the supernatant, the cells were suspended in PBS. Theresulting suspension was centrifuged again (1000 rpm, 5 minutes). Afterthe supernatant was removed, the residue was adjusted to 5×10⁷ cells/mlwith PBS.

2-3) Habituation of Mice

In the inspection and habituation period of 16 days, solid feed anddrinking water were given freely.

2-4) Inoculation with the Cancer Cells

After the inspection and habituation were finished, hair of the animalswas shaved by using hair clippers. Said cells (100 μL/site, 5×10⁶cells/body) were administered intracutaneously into the backs of 59 miceby using a disposal syringe and needle (26G). On the next day ofadministration, the cells were administered in the same manner into 57animals (animals not administered).

2-5) Grouping of the Animals

The tumor diameter (major axis, minor axis) was measured on Days 4, 5, 6and 7 after transplantation (measurement was not conducted aftergrouping). Animals with a tumor diameter (major axis) of 4.5 to 5.5 mm(actual measurement of 4.64 to 5.48 mm) were grouped by stratifiedrandomization such that the average tumor diameter (major axis) becamealmost the same in each group.

2-6) Administration

Using a disposal syringe and needle, the control substance (1000 μL) wasonce administered intraperitoneally into each group, and 1 day later,the test substance (100 μL) was administered into the tumor.

2-7) Measurement of Tumor Diameter

The administration day was regarded as Day 0 after administration. OnDays 3, 6, 9, 12, 15, 18 and 21 after administration, the tumordiameters in each mouse was measured and the tumor volume (majoraxis×minor axis×minor axis÷2) was calculated.

2-8) Measurement of Tumor Weight

All mice in each group (after fasting for 16 to 24 hours) on Day 21after administration were subjected to euthanasia by exsanguinationunder anesthesia by intraperitoneal administration of an aqueoussolution (6.48 mg/mL, 5 mL/kg) of pentobarbital sodium (Tokyo KaseiKogyo Co., Ltd.), and then the tumor was excised and measured for itsweight.

(3) Results

3-1) Tumor Volume

In the control group, HVJ-E group, 13 mg/tumor HVJ-E/BLM group, 0.2mg/body CDDP group, 0.4 mg/body CDDP group and 0.2 mg/body CDDP-13mg/tumor HVJ-E/BLM group, the tumor volume was increased with time untilDay 21 after administration, and the average tumor volumes on Day 21were 3216.14, 2716.00, 2283.84, 1720.14, 1367.62 and 1022.34 mm³respectively in the above groups (3 mice in the 0.4 mg/body CDDP group,and 10 mice in each of the other groups). In one surviving mouse in the0.4 mg/body CDDP-HVJ-E group, the tumor volume on Day 6 afteradministration was increased to 122.27 mm³, and the tumor volume wasdecreased to 118.82 mm³ on Day 9 after administration, and further to13.12-23.26 mm³ on Day 12 to Day 21 after administration (see FIG. 4).

3-2) Tumor Weight

The tumor weights on Day 21 after administration were in the order ofthe control group (2570.35 mm)≧HVJ-E group (2428.64 mg)>13 mg/tumorHVJ-E/BLM group (1680.65 mg)≧0.2 mg/body CDDP group (1619.79 mg)>0.4mg/body CDDP group (1164.13 mg)>0.2 mg/body CDDP-13 mg/tumor HVJ-E/BLMgroup (987.33 mg)>0.4 mg/body CDDP-HVJ-E group (90.4 mg). In the 0.2mg/body CDDP-13 mg/tumor HVJ-E/BLM group, the tumor present at the timeof administration disappeared in 2 out of 10 mice.

The foregoing results revealed that the 13 mg/tumor HVJ-E/BLM groupshows an antitumor action on the CT-26 cells transplanted in mice, andits action is enhanced by combined use of intraperitoneal administrationof CDDP.

1. A pharmaceutical preparation comprising, as an active ingredient, aSendai virus envelope vector having a chemotherapeutic agentincorporated therein.
 2. The pharmaceutical preparation according toclaim 1, wherein the chemotherapeutic agent is a cancerocidal agent, ananticancer agent, or an antitumor agent.
 3. A pharmaceutical preparationcomprising, as an active ingredient, a Sendai virus envelope vectorhaving a chemotherapeutic agent incorporated therein; wherein thechemotherapeutic agent is at least one member selected from the groupconsisting of bleomycin and derivatives thereof, anthraquinone-basedcancerocidal agents, mitomycin and derivatives thereof, actinomycin andderivatives thereof, taxane derivatives, camptothecin and derivativesthereof, cisplatin and derivatives thereof, staurosporine andderivatives thereof, vincristine, streptozotocin, 5-fluorouracil (5-FU)and derivatives thereof, viralbicin, dolastatin, and pharmacologicallyacceptable salts thereof.
 4. The pharmaceutical preparation according toclaim 3, wherein the chemotherapeutic agent is bleomycin or apharmacologically acceptable salt thereof or peplomycin or apharmacologically acceptable salt thereof.
 5. The pharmaceuticalpreparation according to claim 3, wherein the chemotherapeutic agent isat least one member selected from the group consisting of bleomycinhydrochloride, bleomycin sulfate and peplomycin sulfate.
 6. Thepharmaceutical preparation according to claim 1, which is an injection.7. A method of making a pharmaceutical preparation, which comprises:incorporating a chemotherapeutic agent into a Sendai virus envelopevector using a surfactant.
 8. The method of making a pharmaceuticalpreparation according to claim 7, wherein the surfactant is one memberselected from the group consisting of Triton X100, deoxycholic acid andsalts thereof, and cholic acid and salts thereof.
 9. A method oftreating a solid cancer, which comprises: administering to a patient apharmaceutical preparation comprising, as an active ingredient. a Sendaivirus envelope vector having a chemotherapeutic agent for the solidcancer incorporated therein.
 10. The method of treating a solid canceraccording to claim 9, wherein the solid tumor is one member selectedfrom the group consisting of lung cancer, breast cancer, digestive organcancer, head and neck cancer, gynecologic cancer, urologic cancers, softtissue and bone sarcoma, malignant lymphoma and cancer of unknownprimary.
 11. The method of treating a solid cancer according to claim 9,wherein the solid tumor is one member selected from the group consistingof stomach cancer, colon cancer and esophagus cancer.
 12. The method oftreating a solid cancer according to claim 9, wherein the solid tumor isone member selected from the group consisting of upper jaw cancer,tongue cancer, lip cancer, pharynx cancer, larynx cancer and oral cavitycancer.
 13. The method of treating a solid cancer according to claim 9,wherein the solid tumor one member selected from the group consisting ofuterus cancer, ovarian cancer and uterine cervical cancer.
 14. Themethod of treating a solid cancer according to claim 9, wherein thesolid tumor prostate cancer.
 15. A method of treating a cancer, whichcomprises using a chemotherapeutic agent-incorporated Sendai virusenvelope vector in combination with a platinum complex and/or anantimetabolite.
 16. The method of treating a cancer according to claim15, wherein the platinum complex is one member selected from the groupconsisting of cisplatin, carboplatin, Parapratin and nedaplatin.
 17. Themethod of treating a cancer according to claim 15, wherein theantimetabolite is one member selected from the group consisting of6-mercaptopurine riboside, enocitabin, gemcitabine HOl, carmofur,cytarabine, cytarabine ocfosfate, tegafur, tegafur-uracil,tegafur-gimeracil-oteracil-potassium, doxifluridine, hydroxycarbamide,fluorouracil, methotrexate, mercaptopurine and fludarabine phosphate.18. A method of treating a cancer, which comprises using achemotherapeutic agent-incorporated Sendai virus envelope vector incombination with cisplatin and/or fluorouracil.
 19. The method oftreating a cancer according to claim 18, which comprises using achemotherapeutic agent-incorporated Sendai virus envelope vector incombination with cisplatin and/or fluorouracil, and subsequentirradiation.
 20. The method of treating a cancer according to claim 18,which comprises using a bleomycin or its pharmacologically acceptablesalt-incorporated Sendai virus envelope vector in combination withcisplatin and/or fluorouracil, and subsequent irradiation.
 21. Themethod of treating a solid cancer according to claim 9, wherein thepharmaceutical preparation is administered by an injection.
 22. Themethod of treating a solid cancer according to claim 9, wherein thechemotherapeutic agent is at least one member selected from the groupconsisting of bleomycin and derivatives thereof, anthraquinone-basedcancerocidal agents, mitomycin and derivatives thereof, actinomycin andderivatives thereof, taxane derivatives, camptothecin and derivativesthereof, cisplatin and derivatives thereof, staurosporine andderivatives thereof, vincristine, streptozotocin, 5-fluorouracil (5-FU)and derivatives thereof, viralbicin, dolastatin, and pharmacologicallyacceptable salts thereof.
 23. The method of treating a solid canceraccording to claim 9, wherein the chemotherapeutic agent is at least onemember selected from the group consisting of bleomycin hydrochloride,bleomycin sulfate and peplomycin sulfate.